Vaccines comprising mutant attenuated influenza viruses

ABSTRACT

The invention provides a vaccine comprising an effective amount of an isolated recombinant influenza virus comprising a mutant M gene segment that is mutated so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and either lacking a transmembrane domain or having a mutated transmembrane domain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. application Ser. No. 61/316,564, filed on Mar. 23, 2010, the disclosure of which is incorporated by reference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under AI047446 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Influenza A viruses cause a highly contagious, acute respiratory disease responsible for human suffering and economic burden every winter. Vaccination is a primary means for prophylaxis against influenza infection, and inactivated and live attenuated influenza virus vaccines are currently available. Inactivated vaccines, administered parenterally, are generally 70% to 90% effective for reducing the incidence of clinical illness in healthy persons as long as the antigenicities of the circulating virus strains match those of the vaccine (Cox et al., 1999). However, because mucosal immunity and cytotoxic T-cell responses are limited, protective efficacy of inactivated vaccines lasts for only a short period, requiring annual vaccination. In contrast, live attenuated influenza virus vaccines are intranasally administered and elicit robust mucosal immunity and cellular responses; their protective efficacy, therefore, lasts for longer periods (Cox et al., 2004). Only two live attenuated vaccines are currently on the market, and use of these vaccines in the United States is limited to persons aged 2 to 49 years (CDC, 2007).

Generally, influenza vaccines have been prepared from live, attenuated virus or killed virus which can grow to high titers. Live virus vaccines activate all phases of the immune system and stimulate an immune response to each of the protective antigens, which obviates difficulties in the selective destruction of protective antigens that may occur during preparation of inactivated vaccines. In addition, the immunity produced by live virus vaccines is generally more durable, more effective, and more cross-reactive than that induced by inactivated vaccines. Further, live virus vaccines are less costly to produce than inactivated virus vaccines. However, the mutations in attenuated virus are often ill-defined.

For the existing seasonal human influenza, both inactivated virus vaccine and live attenuated virus vaccine are available. In April 2007, the U.S. Food and Drug Administration (FDA) announced the first approval of an inactivated vaccine for humans against the H5N1 virus. However, the available data indicate that inactivated H5 influenza vaccines are suboptimal in their immunogenicity, and a large amount of hemagglutinin (HA) glycoprotein or coadministration of an adjuvant is required to achieve an adequate immune response (Bressen et al., 2006; Lin et al., 2006; Nicholson et al.; 2005; Stephenson et al., 2003; Treanor et al.; 2006).

SUMMARY OF THE INVENTION

The wild-type influenza A virus M2 protein consists of three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain (Lamb et al., 1985; Zebedee et al., 1985). The M2 transmembrane domain has ion channel activity, which functions at an early stage of the viral life cycle between the steps of virus penetration and uncoating (Helenius, 1992; Pinto et al., 1992). The M2 cytoplasmic tail domain has an important role in viral assembly and morphogenesis (Iwatsuki-Horimoto et al., 2006; McCown et al., 2006; McCown et al., 2005). The M2 protein is encoded by a gene segment (the M gene segment) that also encodes the M1 protein, which forms a viral core having viral ribonucleic acid-nucleoprotein complexes. M1 protein and M2 protein share N-terminal sequences. The M2 protein is encoded by a spliced transcript and RNAs encoding the M1 protein and the M2 protein share 3′ sequences, although the coding sequences for M1 and M2 in those 3′ sequences are in different reading frames. The C-terminal residues of M1 and C-terminal portion of the extracellular domain of M2 are encoded by the overlapping 3′ coding sequences.

The invention provides a vaccine comprising an effective amount of an isolated recombinant influenza virus comprising a mutant M gene segment that is mutated so that upon viral replication, the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and a deletion of at least a portion of the transmembrane domain, e.g., internal or C-terminal deletions, and/or includes one or more substitutions in the transmembrane domain. In one embodiment, the mutant M2 protein has a deletion that includes the entire cytoplasmic tail and transmembrane domain of M2, and has one or more residues of the extracellular domain, e.g., has the first 9 to 15 residues of the extracellular domain. The replication of the recombinant virus is attenuated in vivo relative to a corresponding virus without a mutant M gene segment. The recombinant influenza virus of the invention replicates in vitro in the presence of M2 supplied in trans, e.g., producing titers that are substantially the same or at most 10, 100 or 1,000 fold less than a corresponding wild-type influenza virus.

A “functional” M1 protein provides for export of viral nucleic acid from the host cell nucleus, a viral coat, and/or virus assembly and budding. Thus, the M1 protein in the recombinant influenza viruses of the invention has substantially the same function (e.g., at least 10%, 20%, 50% or greater) as a wild-type M1 protein. Thus, any alteration in the M1 coding region in a mutant M gene segment in a recombinant influenza virus does not substantially alter the replication of that virus, e.g., in vitro, for instance, viral titers are not reduced more than about 1 to 2 logs in a host cell that supplies M2 in trans.

As described hereinbelow, an influenza virus with a M gene segment encoding a M2 protein having a deletion of the transmembrane and cytoplasmic domains was prepared in a cell line than expresses M2 in trans. The resulting virus (a M2 “knockout”) was infectious, which was surprising as M2 was believed to be critical for the viral life cycle. In addition, the recombinant viruses of the invention unexpectedly produced progeny viruses, e.g., about 10² pfu/mL in cells that did not provide M2 in trans. The virus of the invention is safer than other attenuated viruses because its reversion rate would be expected to be low and it is highly attenuated, as shown by reduced replication in lung and undetectable replication in nasal turbinates (the virus has greatly reduced capacity to replicate in “normal” cells). Moreover, it was surprising that the cytoplasmic tail of M2 was not necessary in vivo and that such a highly attenuated virus was so immunogenic, e.g., provided protective efficacy.

In one embodiment, the live attenuated influenza virus of the invention elicits both systemic and mucosal immunity at the primary portal of infection. In one embodiment, the live, attenuated influenza virus of the invention has reduced replication in lung compared to wild-type influenza virus, e.g., the live, attenuated influenza virus has titers in lung that are at least one to two logs less, and in one embodiment, replication in nasal turbinates is not detectable. The live, attenuated virus may be employed in a vaccine or immunogenic composition, and so is useful to immunize a vertebrate, e.g., an avian or a mammal, or induce an immune response in a vertebrate, respectively.

In one embodiment, the mutations in the M2 gene result in a mutant M2 protein with a deletion of the entire cytoplasmic tail and deletion or substitution of one or more residues in the transmembrane (TM) domain of M2 and may also comprise at least one amino acid substitution in the extracellular domain, or a combination thereof, relative to a corresponding wild-type M2 protein encoded by a M gene segment. For example, substitutions in the TM domain may include those at residues 25 to 43 of M2, e.g., positions 27, 30, 31, 34, 38, and/or 41 of the TM domain of M2. Substitutions and/or deletions in the TM domain may result in a truncated M2 protein that is not embedded in the viral envelope. For example, a deletion of 10 residues at the C-terminus of the transmembrane domain may result in a truncated M2 protein that is not embedded in the viral envelope. In another embodiment of the invention, the mutant M2 protein may also comprise a deletion in at least a portion of the extracellular domain in addition to deletion of the cytoplasmic domain and a deletion in the TM domain. In one embodiment, the mutant M2 protein has a deletion of the entire cytoplasmic tail and the TM domain and at least one residue of the extracellular domain, e.g., 1 to 15 residues, or any integer in between, of the C-terminal portion of the extracellular domain. In yet another embodiment of the invention, the mutant M2 protein having at least a portion of the extracellular domain further comprises a heterologous protein, e.g., the cytoplasmic and/or TM domain of a heterologous protein (a non-influenza viral protein), which may have a detectable phenotype, that is fused to the C-terminus of at least the extracellular domain of M2, forming a chimeric protein. In one embodiment, the presence of one or more substitutions, deletions, or insertions of heterologous sequences, or any combination thereof, in the M2 gene does not substantially alter the properties of the recombinant influenza virus of the invention, e.g., the presence of one or more substitutions, deletions, or insertions of heterologous sequences does not result in virus titers in vitro that are more than about 1.5 to 2 logs lower, and/or does not result in virus that is substantially less attenuated in vivo, than the recombinant influenza virus of the invention with a mutant M2 protein gene having a deletion of the cytoplasmic tail and TM domain of M2.

In one embodiment, the deletion in the TM domain of M2 includes 1, 2, 3, 4, 5 or more, e.g., 11, 12, 13, 14, or 15 residues, up to 19 residues. In one embodiment, the deletion is from 2 up to 9 residues, including any integer in between. In one embodiment, the deletion is from 15 up to 19 residues, including any integer in between. In one embodiment, the deletion is from 10 up to 19 residues, including any integer in between. In one embodiment, the deletion is the result of at least one substitution of a codon for an amino acid to a stop codon. In one embodiment, the deletion is the result of deletion of at least one codon for an amino acid. In one embodiment, the TM domain of M2 has one or more substitutions, e.g., includes 1, 2, 3, 4, 5 or more, e.g., 11, 12, 13, 14, or 15 substitutions, up to 19 residues of the TM domain. In one embodiment, the one or more amino acid deletions and/or substitutions in the TM domain in a mutant M2 protein that also lacks the cytoplasmic tail of M2, provides for a mutant M2 protein that lacks M2 activity and/or when expressed in a virus yields a live, attenuated virus.

In one embodiment, a deletion in the extracellular (ectodomain) domain of M2 may include 1, 2, 3, 4 or more, e.g., 5, 10, 15, or 20 residues, up to 24 residues of the extracellular domain. In one embodiment, the deletion in the extracellular domain is from 1 up to 15 residues, including any integer in between. In one embodiment, the deletion is the result of at least one substitution of a codon for an amino acid to a stop codon. In one embodiment, the deletion is the result of deletion of at least codon for an amino acid. In one embodiment, the extracellular domain of M2 may also include one or more substitutions. In one embodiment, the mutations in the M2 gene of a M gene segment that result in deletion(s) or substitution(s) in the extracellular domain of M2 do not substantially alter the function of the protein encoded by the M1 gene.

In one embodiment of the invention, fewer than 20%, e.g., 10% or 5%, of the residues in the TM domain or extracellular domain are substituted. In one embodiment, fewer than 60%, e.g., 50%, 40%, 30%, 20%, 10%, or 5% of the residues in the extracellular domain are deleted. In one embodiment, more than 20%, e.g., 30%, 40%, 50%, 80% or more, of the residues in the TM domain are deleted.

Also provided is a method of preparing a recombinant influenza virus comprising a mutant so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and lacking a TM domain or having a mutated TM domain that is truncated and/or includes one or more substitutions, wherein the replication of the recombinant virus is attenuated in vivo relative to a corresponding virus without a mutant M gene. In one embodiment, the virus of the invention may be prepared by mutating a M gene segment. For example, the coding region for the C-terminus of the M1 protein may be mutated by substituting a codon for one, or for two or more adjacent amino acids, in the about 15 C-terminal terminal residues, with one or more stop codons which optionally also substitutes a stop codon in the coding region for the extracellular domain of the M2 protein. The method comprises contacting a host cell with a plurality of influenza vectors, including a vector comprising the mutant M2 sequence, so as to yield recombinant virus. For example, the host cell is contacted with vectors for vRNA production including a vector comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS DNA linked to a transcription termination sequence, wherein the M DNA comprises mutant M2 DNA for a M2 so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and lacking a TM domain or having a mutated TM domain that is truncated and/or includes one or more substitutions; and vectors for mRNA (protein) production including a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally one or more of a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter operably linked to a DNA segment encoding a M2 protein, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS. The host cell may stably express M2, or may be induced to stably express M2. In one embodiment, separate vectors for M1 and M2 vRNA, and/or for NS1 and NS2 vRNA, in place of vectors for M vRNA and/or NS vRNA, are provided and employed. In one embodiment, the promoter in a vRNA vector includes but is not limited to a RNA polymerase I (PolI) promoter, e.g., a human RNA PolI promoter, a RNA polymerase II (PolII) promoter, a RNA polymerase III promoter, a SP6 promoter, a T7 promoter, or a T3 promoter. In one embodiment, one or more vRNA vectors include a PolII promoter and ribozyme sequences 5′ to influenza virus sequences and the same or different ribozyme sequences 3′ to the influenza virus sequences. In one embodiment, the mutant M2 gene is in a vector and is operably linked to a promoter including, but not limited to, a RNA PolI promoter, e.g., a human RNA PolI promoter, a RNA PolII promoter, a RNA polymerase III promoter, a SP6 promoter, a T7 promoter, or a T3 promoter. In one embodiment, the vRNA vectors include a transcription termination sequence including, but not limited to, a PolI transcription termination sequence, a PolII transcription termination sequence, or a PolIII transcription termination sequence, or one or more ribozymes. In one embodiment, the host cell is not contacted with the NA vector, and the resulting virus is further attenuated. In one embodiment, one or more vectors for vRNA production are on the same plasmid (see, e.g., U.S. published application No. 20060166321, the disclosure of which is incorporated by reference herein). In one embodiment, one or more vectors for mRNA production are on the same plasmid (see, e.g., U.S. published application No. 2006/0166321).

In another embodiment, the method includes contacting a host cell with a vector having a PolII promoter linked to a PolI transcription termination sequence linked to an influenza virus PA DNA linked to a PolI promoter linked to a PolI transcription termination sequence (a bidirectional cassette), a vector having a PolII promoter linked to a PolI transcription termination sequence linked to an influenza virus PB1 DNA linked to a PolI promoter linked to a PolII transcription termination sequence, a vector having a PolII promoter linked to a PolI transcription termination sequence linked to an influenza virus PB2 DNA linked to a PolII promoter linked to a PolII transcription termination sequence, a vector having a PolII promoter linked to a PolI transcription termination sequence linked to an influenza virus HA DNA linked to a PolI promoter linked to a PolII transcription termination sequence, a vector having a PolII promoter linked to a PolI transcription termination sequence linked to an influenza virus NP DNA linked to a PolI promoter linked to a PolII transcription termination sequence, a vector having a PolII promoter linked to a PolI transcription termination sequence linked to an influenza virus NA DNA linked to a PolI promoter linked to a PolII transcription termination sequence, a vector having a PolII promoter linked to a PolI transcription termination sequence linked to an influenza virus M DNA linked to a PolI promoter linked to PolII transcription termination sequence, and a vector having a PolI promoter linked to a PolI transcription termination sequence linked to an influenza virus NS DNA linked to a PolI promoter linked to PolII transcription termination sequence, wherein the M DNA comprises mutant M2 DNA for a M2 so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and lacking a TM domain or having a mutated TM domain that is truncated and/or includes one or more substitutions, wherein the replication of the recombinant virus is attenuated in vivo relative to a corresponding virus without a mutant M gene segment. The host cell may stably express M2, or may be induced to stably express M2.

Also provided is a method of preparing a recombinant influenza virus comprising a mutant M2 gene for a M2 so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and lacking a TM domain or having a mutated TM domain that is truncated and/or includes one or more substitutions, wherein the replication of the recombinant virus is attenuated in vivo relative to a corresponding virus without the mutant M gene segment. The method comprises contacting a host cell with a plurality of influenza vectors, including a vector comprising a PolI promoter operably linked to an influenza virus PA DNA linked to a PolI transcription termination sequence, a vector comprising a PolI promoter operably linked to an influenza virus PB1 DNA linked to a PolI transcription termination sequence, a vector comprising a PolI promoter operably linked to an influenza virus PB2 DNA linked to a PolI transcription termination sequence, a vector comprising a PolI promoter operably linked to an influenza virus HA DNA linked to a PolI transcription termination sequence, a vector comprising PolI promoter operably linked to an influenza virus NP DNA linked to a PolI transcription termination sequence, a vector comprising a PolI promoter operably linked to an influenza virus DNA NA linked to a PolI transcription termination sequence, a vector comprising a PolI promoter operably linked to an influenza virus M DNA linked to a PolI transcription termination sequence, and a vector comprising a PolI promoter operably linked to an influenza virus NS DNA linked to a PolI transcription termination sequence. The sequence of the DNA for M comprises a M2 sequence for a mutant M2 so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and lacking a TM domain or having a mutated TM domain that is truncated and/or includes one or more substitutions, wherein the replication of the recombinant virus is attenuated in vivo relative to a corresponding virus without the mutant M gene segment; and a vector comprising a PolII promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a PolII promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a PolII promoter operably linked to a DNA segment encoding influenza virus PB2, a vector comprising a PolII promoter operably linked to a DNA segment encoding influenza virus NP, and optionally one or more of a vector comprising a PolII promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a PolII promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a PolII promoter operably linked to a DNA segment encoding influenza virus M and a vector comprising a PolII promoter operably linked to a DNA segment encoding influenza virus NS. The host cell may stably express M2, or may be induced to stably express M2. In one embodiment, separate vectors for M1 and M2 vRNA, and/or for NS1 and NS2 vRNA, in place of vectors for M vRNA and/or NS vRNA, are provided and employed. In one embodiment, the PolI promoter is a human PolI promoter. In one embodiment, the PolI promoter for each PolI containing vector is the same. In one embodiment, the PolII promoter for each PolII containing vector is the same. In one embodiment, the PolII promoter for two or more, but not all, of the PolII containing vectors, is the same. In one embodiment, the PolII promoter for each PolII containing vector is different.

The invention further provides a composition having one or more of the vectors described above, and a host cell contacted with such a composition, e.g., so as to yield infectious virus. The host cell may be contacted with each vector, or a subset of vectors, sequentially. One or more of the vectors may be on plasmids. The compositions and host cells of the invention may also include another vector for vRNA production or protein production that includes heterologous sequences, e.g., for a marker gene, or a therapeutic or prophylactic gene, e.g., an immunogen for a cancer associated antigen or for a pathogen such as a bacteria, a noninfluenza virus, fungus, or other pathogen.

In one embodiment, the recombinant virus of the invention includes one or more genes from influenza A virus. In another embodiment, the recombinant virus of the invention may include one or more genes from influenza B virus, e.g., an influenza B HA gene. In yet another embodiment, the recombinant virus of the invention may include one or more genes from influenza C virus.

In one embodiment, the influenza DNA in a vector is a DNA with a native (naturally occurring) influenza virus sequence. In one embodiment, the influenza DNA is a DNA that has been manipulated in vitro, e.g., by inserting, deleting or substituting, or a combination thereof, one or more nucleotides in, for example, the coding region.

The HA sequences in a recombinant virus of the invention may be any one of the sixteen influenza A HA sequences, a chimeric HA sequence or any non-native HA sequence. The NA sequences in a recombinant virus of the invention may be any one of the nine influenza A NA sequences, a chimeric NA sequence or any non-native NA sequence.

In one embodiment, other attenuating mutations may be introduced to the vectors, e.g., a mutation in a HA cleavage site that results in a site that is not cleaved.

Further provided is a receptacle containing a composition comprising one or more influenza viruses, at least one of which is a live, attenuated influenza virus of the invention, each in an amount effective to provide a protective immune response. In one embodiment, the composition is formulated for intranasal delivery.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of M2 mutants. The M gene was derived from a highly pathogenic H5N1 (VN1203) virus. The mutants del5, del11, del22, del33, and del44 contain a 5-, 11-, 22-, 33-, or 44-amino-acid (aa) deletion from the C-terminus, respectively. The mutant delM2 was constructed by deletion of 70 C-terminal residues, including the entire TM and cytoplasmic domains.

FIG. 2. Growth kinetics of the M2 tail deletion mutant viruses in MDCK cells. MDCK cells were infected with the M2 tail deletion mutant viruses at an MOI of 0.001. At the indicated times after infection, the virus titer in the supernatant was determined with M2CK cells. The values presented are means from duplicate experiments. WT, wild-type. The del5 and del11 mutants grew as well as the wild-type virus, whereas the del22, del33, del44, and delM2 replicated less efficiently than did the wild-type virus in MDCK cells (about 1,000 to about 10,000-fold lower).

FIG. 3. Pathogenicity of a recombinant M2del11-HAavir virus. Mice were infected with 100, 1,000, 10,000, or 100,000 PFU of the M2del11-HAavir virus, and their body weights were monitored for 14 days. Data are reported as the mean changes in body weight±standard deviation (n=3).

FIG. 4. Virus-specific serum and mucosal antibody responses in mice immunized with the M2del11-HAavir virus. Mice were immunized with 100 or 1,000 PFU of M2del11-HAavir virus intranasally. Samples from each group were obtained 3 weeks postimmunization. IgG and IgA levels in sera (A), lung washes (B), and nasal washes (C) from individual mice were detected by ELISA. Values are expressed as the mean absorbance±standard deviation (n=4) of undiluted samples (trachea-lung and nasal washes) or of samples diluted 1:10 (sera). Differences between responses to PBS and the M2del11-HA virus were tested for statistical significance by the use of Student's t test. M2del11-HAavir showed substantial levels of virus-specific IgG titers in serum and lung wash as well as IgA titers in lung wash, which increased with the immunization dose. These data indicate that M2del11-HAavir was able to induce strong antibody responses in mice.

FIG. 5. Trypsin dependence of plaque formation of M2del11-HAavir virus in M2CK cells. Plaque assays were performed on M2CK cells in the presence or absence of trypsin. M2del11 virus was able to form plaques in both the presence and absence of trypsin. In contrast, with M2del11-HAavir mutant virus, clear plaques were visible only in the presence of trypsin.

FIG. 6. Protection against challenge with lethal doses of H5 viruses of mice immunized with M2del11-HAavir virus. One month after immunization of mice with M2del11-HA, the immunized mice virus survived a lethal challenge with 100 MLD₅₀ of highly pathogenic H5N1 viruses (VN1203 or Indonesia 7 virus) and did not show any symptom (i.e., weight loss) after challenge, whereas all of the control mice died or had to be euthanized due to their disease by day 8 post-challenge.

FIG. 7. Construction of the mutant M segment, growth kinetics of M2KO virus, and change in body weight of mice infected with M2KO virus. (A) Schematic diagram of the mutated M segment of the M2KO virus. Blue and yellow columns represent the open reading frame of the M1 and M2 proteins, respectively. Two stop codons (TGA TGA) were introduced downstream of the open reading frame of the M1 protein in the M segment to eliminate the TM and cytoplasmic tail domains of the M2 protein. M2 ect., M2 TM, and A (n) denote the ectodomain domain of M2, the TM domain of M2, and the poly(A) tail, respectively. Numbers refer to the nucleotide numbers from the 5′ end of the cRNA. (B) Growth properties of PR8 and M2KO viruses in MDCK and M2-expressing MDCK (M2CK) cells. MDCK and M2CK cells were infected with PR8 or M2KO virus at a multiplicity of infection of 0.001. Virus titers in the supernatant of MDCK (left) and M2CK (right) cells at various time points postinfection were determined by using M2CK cells. The dotted line indicates the detection limit of virus titer (10 PFU/ml). (C) Body weight changes in mice inoculated with PBS, PR8, or M2KO virus. The body weights of the control (PBS) or infected (PR8 or M2KO virus) mice were measured daily postinfection. Values are expressed as the mean change in body weight±standard deviations (n=8 for PBS and M2KO virus; n=3 for PR8).

FIG. 8. Virus-specific antibodies in trachea/lung and nasal washes and in sera of mice inoculated with M2KO virus. Samples from each group were obtained 4 weeks postimmunization. Samples were serially diluted, and IgG and IgA in samples from individual mice were detected by use of an enzyme-linked immunosorbent assay. Values are expressed as mean absorbances (n=4).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “isolated and/or purified” refer to in vitro preparation, isolation and/or purification of a nucleic acid molecule such as a vector, plasmid of the invention or a virus of the invention, so that it is not associated with in vivo substances, or is substantially purified from in vitro substances. An isolated virus preparation is generally obtained by in vitro culture and propagation and is substantially free from other infectious agents. As used herein, “substantially free” means below the level of detection for a particular infectious agent using standard detection methods for that agent. A “recombinant” virus is one which has been manipulated in vitro, e.g., using recombinant DNA techniques, to introduce changes to the viral genome, or otherwise artificially generated.

As used herein, the term “recombinant nucleic acid” or “recombinant DNA sequence or segment” refers to a nucleic acid, e.g., to DNA, that has been derived or isolated from a source, that may be subsequently chemically altered in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in the native genome. An example of DNA “derived” from a source, would be a DNA sequence that is identified as a useful fragment, and which is then chemically synthesized in essentially pure form. An example of such DNA “isolated” from a source would be a useful DNA sequence that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.

Influenza Virus

The life cycle of viruses generally involves attachment to cell surface receptors, entry into the cell and uncoating of the viral nucleic acid, followed by replication of the viral genes inside the cell. After the synthesis of new copies of viral proteins and genes, these components assemble into progeny virus particles, which then exit the cell. Different viral proteins play a role in each of these steps.

The influenza A virus is an enveloped negative-strand virus with eight RNA segments encapsidated with nucleoprotein (NP). The eight single-stranded negative-sense viral RNAs (vRNAs) encode a total of ten to eleven proteins. The influenza virus life cycle begins with binding of the hemagglutinin (HA) to sialic acid-containing receptors on the surface of the host cell, followed by receptor-mediated endocytosis. The low pH in late endosomes triggers a conformational shift in the HA, thereby exposing the N-terminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide initiates the fusion of the viral and endosomal membrane, and the matrix protein (M1) and RNP complexes are released into the cytoplasm. RNPs consist of the nucleoprotein (NP), which encapsidates vRNA, and the viral polymerase complex, which is formed by the PA, PB1, and PB2 proteins. RNPs are transported into the nucleus, where transcription and replication take place. The RNA polymerase complex catalyzes three different reactions: synthesis of an mRNA with a 5′ cap and 3′ polyA structure, of a full-length complementary RNA (cRNA), and of genomic vRNA using the cDNA as a template. Newly synthesized vRNAs, NP, and polymerase proteins are then assembled into RNPs, exported from the nucleus, and transported to the plasma membrane, where budding of progeny virus particles occurs. The neuraminidase (NA) protein plays a crucial role late in infection by removing sialic acid from sialyloligosaccharides, thus releasing newly assembled virions from the cell surface and preventing the self aggregation of virus particles. Although virus assembly involves protein-protein and protein-vRNA interactions, the nature of these interactions is largely unknown.

Although influenza B and C viruses are structurally and functionally similar to influenza A virus, there are some differences. For example, the M segment of influenza B virus encodes two proteins, M1 and BM2, through a termination-reinitiation scheme of tandem cistrons, and the NA segment encodes the NA and NB proteins from a bicistronic mRNA. Influenza C virus, which has 7 vRNA segments, relies on spliced transcripts to produce M1 protein; the product of the unspliced mRNA is proteolytically cleaved to yield the CM2 protein. In addition, influenza C virus encodes a HA-esterase (HEF) rather than individual HA and NA proteins.

Spanning the viral membrane for influenza A virus are three proteins: hemagglutinin (HA), neuraminidase (NA), and M2. The extracellular domains (ectodomains) of HA and NA are quite variable, while the ectodomain domain of M2 is essentially invariant among influenza A viruses. The M2 protein which possesses ion channel activity (Pinto et al., 1992), is thought to function at an early state in the viral life cycle between host cell penetration and uncoating of viral RNA (Martin and Helenius, 1991; reviewed by Helenius, 1992; Sugrue et al., 1990). Once virions have undergone endocytosis, the virion-associated M2 ion channel, a homotetrameric helix bundle, is believed to permit protons to flow from the endo some into the virion interior to disrupt acid-labile M1 protein-ribonucleoprotein complex (RNP) interactions, thereby promoting RNP release into the cytoplasm (reviewed by Helenius, 1992). In addition, among some influenza strains whose HAs are cleaved intracellularly (e.g., A/fowl plagues/Rostock/34), the M2 ion channel is thought to raise the pH of the trans-Golgi network, preventing conformational changes in the HA due to conditions of low pH in this compartment (Hay et al., 1985; Ohuchi et al., 1994; Takeuchi and Lamb, 1994).

Evidence that the M2 protein of influenza virus has ion channel activity was obtained by expressing the protein in oocytes of Xenopus laevis and measuring membrane currents (Pinto et al., 1992; Wang et al., 1993; Holsinger et al., 1994). Specific changes in the M2 protein TM domain altered the kinetics and ion selectivity of the channel, providing strong evidence that the M2 TM domain constitutes the pore of the ion channel (Holsinger et al., 1994). In fact, the M2 TM domain itself can function as an ion channel (Duff and Ashley, 1992). M2 protein ion channel activity is thought to be essential in the life cycle of influenza viruses, because amantadine hydrochloride, which blocks M2 ion channel activity (Hay et al., 1993), inhibits viral replication (Kato and Eggers, 1969; Skehel et al., 1978).

Exemplary Viruses and Methods

The invention provides recombinant influenza viruses useful in vivo. In one embodiment, the invention provides an isolated recombinant influenza virus comprising a mutant M2 gene so that upon viral replication the mutant M gene segment expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and lacking a TM domain or having a mutated TM domain that is truncated and/or includes one or more substitutions, wherein the replication of the recombinant virus is attenuated in vivo relative to a corresponding virus without a mutant M gene segment, wherein the replication of the virus in vitro in the presence of M2 supplied in trans is not substantially altered but the recombinant virus is attenuated in vivo relative to a corresponding virus without the deletion.

In one embodiment, the M2 protein has a deletion in the TM domain of at least 3 up to 19 residues. In one embodiment, the deletion that includes residues 29 to 31. In one embodiment, the deletion is at least 5 up to 19 residues. In another embodiment, the deletion is at least 15 residues. In yet another embodiment, the deletion is at least 10 residues. In another embodiment, the deletion is at least 10 residues. In a further embodiment, the deletion includes the entire TM domain.

In one embodiment, the mutant M2 protein further comprises a heterologous protein at the C-terminus. In one embodiment, the mutant M2 protein further comprises at least one amino acid substitution, e.g., in the TM domain of the M2 protein. The isolated virus may further include another attenuating mutation(s) in addition to the deleted M2 protein.

In one embodiment, the recombinant virus comprises influenza A HA, for instance, H5 HA. In one embodiment, the HA is not H3 HA.

Also provided is a method of preparing a recombinant influenza virus comprising a mutant M2 protein gene. The method includes contacting a host cell with a plurality of influenza vectors so as to yield recombinant influenza virus. The plurality of vectors includes: vectors for vRNA production including a vector comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS DNA linked to a transcription termination sequence, wherein the M DNA comprises mutant M2 DNA for a mutant M2 protein with a deletion of the cytoplasmic tail and lacking a TM domain or having a mutated TM domain that is truncated and/or includes one or more substitutions; and b) vectors for protein production including a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter operably linked to a DNA segment encoding an ion channel protein, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS. Virus which replicates in vitro but is attenuated in vivo is isolated from the host cells. In one embodiment, the vector for vRNA production of HA comprises H5 DNA, e.g., one with a mutant cleavage site associated with reduced virulence. In one embodiment, the promoter in the vectors for vRNA production is a PolI promoter. In one embodiment, the vector for vRNA production of HA comprises influenza A HA DNA.

Further provided are compositions with one or more vectors of the invention. In one embodiment, a composition includes a plurality of influenza vectors, for instance, vectors for vRNA production including a vector comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS DNA linked to a transcription termination sequence, wherein the M DNA comprises mutant M2 DNA so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and lacking a TM domain or having a mutated TM domain that is truncated and/or includes one or more substitutions, wherein the replication of the recombinant virus is attenuated in vivo relative to a corresponding virus without a mutant M gene segment; and vectors for mRNA production including a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter operably linked to a DNA segment encoding an ion channel protein, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS. In one embodiment, the composition further includes a vector comprising a promoter operably linked to a heterologous DNA sequence of interest, e.g., wherein the vector comprises a DNA sequence for an immunogenic polypeptide or peptide of a pathogen or wherein the vector comprises a DNA sequence for a therapeutic protein. In one embodiment, two or more of the vectors for vRNA production are on the same plasmid. In one embodiment, two or more of the vectors for mRNA production are on the same plasmid.

Vaccines

A vaccine of the invention may comprise immunogenic proteins including glycoproteins of any pathogen, e.g., an immunogenic protein from one or more bacteria, viruses, yeast or fungi. Thus, in one embodiment, the influenza viruses of the invention may be vaccine vectors for influenza virus or other viral pathogens including but not limited to lentiviruses such as HIV, hepatitis B virus, hepatitis C virus, herpes viruses such as CMV or HSV or foot and mouth disease virus.

A complete virion vaccine is concentrated by ultrafiltration and then purified by zonal centrifugation or by chromatography. It is inactivated before or after purification using formalin or beta-propiolactone, for instance.

A subunit vaccine comprises purified glycoproteins. Such a vaccine may be prepared as follows: using viral suspensions fragmented by treatment with detergent, the surface antigens are purified, by ultracentrifugation for example. The subunit vaccines thus contain mainly HA protein, and also NA. The detergent used may be cationic detergent for example, such as hexadecyl trimethyl ammonium bromide, an anionic detergent such as ammonium deoxycholate; or a nonionic detergent such as that commercialized under the name TRITON X100. The hemagglutinin may also be isolated after treatment of the virions with a protease such as bromelin, then purified by a method such as that described by Grand and Skehel.

A split vaccine comprises virions which have been subjected to treatment with agents that dissolve lipids. A split vaccine can be prepared as follows: an aqueous suspension of the purified virus obtained as above, inactivated or not, is treated, under stirring, by lipid solvents such as ethyl ether or chloroform, associated with detergents. The dissolution of the viral envelope lipids results in fragmentation of the viral particles. The aqueous phase is recuperated containing the split vaccine, constituted mainly of hemagglutinin and neuraminidase with their original lipid environment removed, and the core or its degradation products. Then the residual infectious particles are inactivated if this has not already been done.

Inactivated Vaccines. Inactivated influenza virus vaccines of the invention are provided by inactivating replicated virus of the invention using known methods, such as, but not limited to, formalin or β-propiolactone treatment. Inactivated vaccine types that can be used in the invention can include whole-virus (WV) vaccines or subvirion (SV) (split) vaccines. The WV vaccine contains intact, inactivated virus, while the SV vaccine contains purified virus disrupted with detergents that solubilize the lipid-containing viral envelope, followed by chemical inactivation of residual virus.

In addition, vaccines that can be used include those containing the isolated HA and NA surface proteins, which are referred to as surface antigen or subunit vaccines. In general, the responses to SV and surface antigen (i.e., purified HA or NA) vaccines are similar. An experimental inactivated WV vaccine containing an NA antigen immunologically related to the epidemic virus and an unrelated HA appears to be less effective than conventional vaccines (Ogra et al., 1977). Inactivated vaccines containing both relevant surface antigens are generally employed.

Live Attenuated Virus Vaccines. Live, attenuated influenza virus vaccines, can also be used for preventing or treating influenza virus infection, according to known method steps. Attenuation may be achieved in a single step by transfer of attenuated genes from an attenuated donor virus to a replicated isolate or reassorted virus according to known methods (see, e.g., Murphy, 1993). Since resistance to influenza A virus is mediated by the development of an immune response to the HA and NA glycoproteins, the genes coding for these surface antigens must come from the circulating wild-type strains. The attenuated genes are derived from the attenuated parent. In this approach, genes that confer attenuation do not code for the HA and NA glycoproteins. Otherwise, these genes could not be transferred to reassortants bearing the surface antigens of the clinical virus isolate.

Many donor viruses have been evaluated for their ability to reproducibly attenuate influenza viruses. As a non-limiting example, the A/Ann Arbor(AA)/6/60 (H2N2) cold adapted (ca) donor virus can be used for attenuated vaccine production. Reassortant progeny are then selected at 25° C. (restrictive for replication of virulent virus), in the presence of an H2N2 antiserum, which inhibits replication of the viruses bearing the surface antigens of the attenuated A/AA/6/60 (H2N2) ca donor virus.

A large series of H1N1 and H3N2 reassortants have been evaluated in humans and found to be satisfactorily: (a) infectious, (b) attenuated for seronegative children and immunologically primed adults, (c) immunogenic and (d) genetically stable. The immunogenicity of the ca reassortants parallels their level of replication. Thus, the acquisition of the six transferable genes of the ca donor virus by new wild-type viruses has reproducibly attenuated these viruses for use in vaccinating susceptible adults and children.

Other attenuating mutations can be introduced into influenza virus genes by site-directed mutagenesis to rescue infectious viruses bearing these mutant genes. Attenuating mutations can be introduced into non-coding regions of the genome, as well as into coding regions. Such attenuating mutations can also be introduced, for example, into the PB2 polymerase gene or the NS gene. Thus, new donor viruses can also be generated bearing attenuating mutations introduced by site-directed mutagenesis, and such new donor viruses can be used in the production of live attenuated reassortant H1N1 and H3N2 vaccine candidates in a manner analogous to that described above for the A/AA/6/60 ca donor virus.

In one embodiment, such attenuated viruses maintain the genes from the virus that encode antigenic determinants substantially similar to those of the original clinical isolates. This is because the purpose of the attenuated vaccine is to provide substantially the same antigenicity as the original clinical isolate of the virus, while at the same time lacking infectivity to the degree that the vaccine causes minimal change of inducing a serious pathogenic condition in the vaccinated mammal.

The virus can thus be attenuated or inactivated, formulated and administered, according to known methods, as a vaccine to induce an immune response in an animal, e.g., a mammal. Methods are well-known in the art for determining whether such attenuated or inactivated vaccines have maintained similar antigenicity to that of the clinical isolate or high growth strain derived therefrom. Such known methods include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantidine); HA and NA activity and inhibition; and DNA screening (such as probe hybridization or PCR) to confirm that donor genes encoding the antigenic determinants (e.g., HA or NA genes) are not present in the attenuated viruses.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention, suitable for inoculation or for parenteral or oral administration, comprise attenuated or inactivated influenza viruses, optionally further comprising sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions can further comprise auxiliary agents or excipients, as known in the art. The composition of the invention is generally presented in the form of individual doses (unit doses).

Conventional vaccines generally contain about 0.1 to 200 μg, for example, 10 to 15 μg, of hemagglutinin from each of the strains entering into their composition. The vaccine forming the main constituent of the vaccine composition of the invention may comprise a virus of type A, B or C, or any combination thereof, for example, at least two of the three types, at least two of different subtypes, at least two of the same type, at least two of the same subtype, or a different isolate(s) or reassortant(s). Human influenza virus type A includes H1N1, H2N2 and H3N2 subtypes.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and/or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

When a composition of the present invention is used for administration to an individual, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. For vaccines, adjuvants, substances which can augment a specific immune response, can be used. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the organism being immunized. Examples of materials suitable for use in vaccine compositions are provided in Osol (1980).

Heterogeneity in a vaccine may be provided by mixing replicated influenza viruses for at least two influenza virus strains, such as 2-50 strains or any range or value therein. Influenza A or B virus strains having a modern antigenic composition are often employed. According to the present invention, vaccines can be provided for variations in a single strain of an influenza virus, using techniques known in the art.

A pharmaceutical composition according to the present invention may further or additionally comprise at least one chemotherapeutic compound, for example, for gene therapy, immunosuppressants, anti-inflammatory agents or immune enhancers, and for vaccines, chemotherapeutics including, but not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumor necrosis factor-alpha, thiosemicarbarzones, methisazone, rifampin, ribavirin, a pyrimidine analog, a purine analog, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, a protease inhibitor, or ganciclovir.

The composition can also contain variable but small quantities of endotoxin-free formaldehyde, and preservatives, which have been found safe and not contributing to undesirable effects in the organism to which the composition is administered.

Pharmaceutical Purposes

The administration of the composition (or the antisera that it elicits) may be for either a “prophylactic” or “therapeutic” purpose. When provided prophylactically, the compositions of the invention which are vaccines, are provided before any symptom of a pathogen infection becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. When provided prophylactically, the gene therapy compositions of the invention, are provided before any symptom of a disease becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate one or more symptoms associated with the disease.

When provided therapeutically, an attenuated or inactivated viral vaccine is provided upon the detection of a symptom of actual infection. The therapeutic administration of the compound(s) serves to attenuate any actual infection. When provided therapeutically, a gene therapy composition is provided upon the detection of a symptom or indication of the disease. The therapeutic administration of the compound(s) serves to attenuate a symptom or indication of that disease.

Thus, an attenuated or inactivated vaccine composition of the present invention may thus be provided either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection. Similarly, for gene therapy, the composition may be provided before any symptom of a disorder or disease is manifested or after one or more symptoms are detected.

A composition is said to be “pharmacologically acceptable” if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. A composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, e.g., enhances at least one primary or secondary humoral or cellular immune response against at least one strain of an infectious influenza virus.

The “protection” provided need not be absolute, i.e., the influenza infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of patients. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of the influenza virus infection.

Pharmaceutical Administration

A composition of the present invention may confer resistance to one or more pathogens, e.g., one or more influenza virus strains, by either passive immunization or active immunization. In active immunization, an inactivated or attenuated live vaccine composition is administered prophylactically to a host (e.g., a mammal), and the host's immune response to the administration protects against infection and/or disease. For passive immunization, the elicited antisera can be recovered and administered to a recipient suspected of having an infection caused by at least one influenza virus strain. A gene therapy composition of the present invention may yield prophylactic or therapeutic levels of the desired gene product by active immunization.

In one embodiment, the vaccine is provided to a mammalian female (at or prior to pregnancy or parturition), under conditions of time and amount sufficient to cause the production of an immune response which serves to protect both the female and the fetus or newborn (via passive incorporation of the antibodies across the placenta or in the mother's milk).

The present invention thus includes methods for preventing or attenuating a disorder or disease, e.g., an infection by at least one strain of pathogen. As used herein, a vaccine is said to prevent or attenuate a disease if its administration results either in the total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or in the total or partial immunity of the individual to the disease. As used herein, a gene therapy composition is said to prevent or attenuate a disease if its administration results either in the total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or in the total or partial immunity of the individual to the disease.

At least one inactivated or attenuated influenza virus, or composition thereof, of the present invention may be administered by any means that achieve the intended purposes, using a pharmaceutical composition as previously described.

For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration can be by bolus injection or by gradual perfusion over time. One mode of using a pharmaceutical composition of the present invention is by intramuscular or subcutaneous application.

A typical regimen for preventing, suppressing, or treating an influenza virus related pathology, comprises administration of an effective amount of a vaccine composition as described herein, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including between one week and about 24 months, or any range or value therein.

According to the present invention, an “effective amount” of a composition is one that is sufficient to achieve a desired biological effect. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect wanted. The ranges of effective doses provided below are not intended to limit the invention and represent exemplary dose ranges. However, the dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art.

The dosage of an attenuated virus vaccine for a mammalian (e.g., human) or avian adult organism can be from about 10³-10⁷ plaque forming units (PFU)/kg, or any range or value therein. The dose of inactivated vaccine can range from about 0.1 to 200, e.g., 50 μg of hemagglutinin protein. However, the dosage should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point.

The dosage of immunoreactive HA in each dose of replicated virus vaccine can be standardized to contain a suitable amount, e.g., 1-50 μg or any range or value therein, or the amount recommended by the U.S. Public Heath Service (PHS), which is usually 15 μg, per component for older children 3 years of age, and 7.5 μg per component for older children <3 years of age. The quantity of NA can also be standardized, however, this glycoprotein can be labile during the processor purification and storage. Each 0.5-ml dose of vaccine may contain approximately 1-50 billion virus particles, and for example 10 billion particles.

The invention will be further described by the following non-limiting examples.

Example 1 Materials and Methods

Cells. 293T human embryonic kidney cells and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and in minimal essential medium (MEM) containing 5% newborn calf serum, respectively. All cells were maintained at 37° C. in 5% CO₂. Hygromycin-resistant MDCK cells stably expressing M2 protein from A/Puerto Rico/8/34 (H1N1) were established by cotransfection with plasmid pRHyg, containing the hygromycin resistance gene, and plasmid pCAGGS/M2, expressing the full-length M2 protein, at a ratio of 1:1. The stable MDCK cell clone (M2CK) expressing M2 was selected in medium containing 0.15 mg/mL of hygromycin (Roche, Mannheim, Germany) by screening with indirect immunostaining using an anti-M2 (14C2) monoclonal antibody. The M2CK cells were cultured in MEM supplemented with 10% fetal calf serum and 0.15 mg/mL of hygromycin. In M2CK cells, the expression levels and localization of M2 were similar to those in virus-infected cells (data not shown).

Plasmid construction. The cDNA of A/Vietnam/1203/04 (VN1203) virus was synthesized by reverse transcription of viral RNA with an oligonucleotide complementary to the conserved 3′ end of the viral RNA, as described by Katz et al. (1990). The cDNA was amplified by PCR with M gene-specific oligonucleotide primers containing BsmBI sites, and PCR products were cloned into the pGEM vector. The resulting construct was designated pGEM-VN1203M. After digestion with BsmBI, the fragment was cloned into the BsmBI sites of the pHH21 vector, which contains the human RNA polymerase I promoter and the mouse RNA polymerase I terminator, separated by BsmBI sites, resulting in pPolIUdM. Plasmids derived from pHH21 for the expression of viral RNA are referred to as “PolI” constructs herein.

The M mutants were constructed as follows. pGEM-VN1203M was first amplified by inverse PCR (Ochmann et al., 1988) using the back-to-back primers M987stopF (5′-gtgaATAGAATTGGAGTAAAAAACTACC-3′; SEQ ID NO:1) and M987stopR (5′-tcaAAAATGACCATCGTCAACATCCAC-3′; SEQ ID NO:2), M969stopF (5′-gtgaGATGGTCATTTTGTCAACATAGAA-3′; SEQ ID NO:3) and M969stopR (5′-tcaATCCACAGCACTCTGCTGTTCCTG-3′; SEQ ID NO:4), M936stopF (5′-gtgaCGGCAGGAACAGCAGAGTGCTG-3′; SEQ ID NO:5) and M936stopR (5′-tcaTTCCCTCATAGACTCAGGTACC-3′; SEQ ID NO:6), M903stopF (5′-gtgaGCAGGGGTACCTGAGTCTATG-3′; SEQ ID NO:7) and M903stopR (5′-tcaAGGCCCTCTTTTCAAACCGTA-3′; SEQ ID NO:8), M870stopF (5′-CTTAAATACGGTTTGAAAAGAGGGCCTGC-3′; SEQ ID NO:9) and M870stopR (5′-tcactcaATAAATGCATTTGAAGAAAAGACGATC-3′; SEQ ID NO:10), and M783stopF (5′-TTGTTGTTGCCGCAAATATCATTGGG-3′; SEQ ID NO:11) and M783stopR (5′-TtcactcaACTTGAATCGCTGCATCTGC-3′; SEQ ID NO:12). Nucleotide changes to introduce stop codons are indicated by lowercase letters.

The PCR products were then phosphorylated, self-ligated, propagated in Escherichia coli strain DH5α, and then digested with BsmBI and cloned into the BsmBI sites of the pHH21 vector. The resulting constructs were designated pPolI-VN1203M2del5, pPolI-VN1203M2del11, pPolI-VN1203M2del22, pPolI-VN1203M2del33, pPolI-VN1203M2del44, and pPolI-VN1203delM2, each of which contained two stop codons at nucleotide positions 972 to 974, 939 to 941, 906 to 908, 873 to 875, and 786 to 788 of the M segment, which resulted in the deletion of 5, 11, 22, 33, 44, and 70 residues from the C-terminus of the M2 protein, respectively (FIG. 1). All of the constructs were sequenced to ensure that unwanted mutations were not present.

Plasmid-driven reverse genetics. All of the viruses were generated by the introduction of plasmids expressing eight viral RNA segments and three polymerase proteins plus NP, as described by Neumann et al. (1999). At 48 hours posttransfection, viruses were harvested and used to inoculate M2CK cells for the production of stock viruses. The M genes of transfectant viruses were sequenced to confirm the origin of the gene and the presence of the intended mutations and to ensure that no unwanted mutations were present. All experiments with live viruses and with transfectants generated by reverse genetics were performed in a biosafety level 3 containment laboratory approved for such use by the CDC and the U.S. Department of Agriculture.

Replicative properties of the transfectant viruses in cell culture. MDCK cells were infected in duplicate wells of 24-well plates with the wild-type or mutant viruses at a multiplicity of infection (MOI) of 0.001, overlaid with MEM containing 0.5 μg of trypsin per mL, and incubated at 37° C. At select time points, supernatants were assayed for infectious virus in plaque assays on M2CK cells (Iwatsuki-Horimoto et al., 2006).

Experimental infection. Five-week-old female BALB/c mice, anesthetized with isoflurane, were infected intranasally with 50 μL (100 PFU) of virus. Virus titers in organs were determined 3 days after infection by use of MDCK cells, as described in Bilsel et al. (1993).

Immunization and protection. BALB/c mice (4-week-old females) were intranasally immunized with 100 or 1,000 PFU/50 μL of the M2del11-HAavir virus. Three weeks later, four mice were sacrificed to obtain sera, trachea-lung washes, and nasal washes. One month after vaccination, immunized mice were challenged intranasally, under anesthesia, with 3.8×10² PFU or 5×10⁴ PFU of the wild-type VN1203 or A/Indonesia/7/05 virus, which was equivalent to 100 50% minimal lethal doses (MLD₅₀) (dose required to kill 50% of infected mice), respectively. To determine virus titers in mice, organ samples were harvested at day 3 postchallenge and were homogenized and titrated on MDCK cells. The remaining animals were observed for clinical signs and symptoms of infection for 14 days postchallenge.

Virus-specific antibody detection. Immunoglobulin G (IgG) and IgA antibody titers were measured in sera, trachea-lung washes, and nasal washes of the immunized mice by use of an enzyme-linked immunosorbent assay (ELISA) (Kida et al., 1982). In this assay, the wells were coated with purified A/Vietnam/1194/05 virus after treatment with 0.05 M Tris-HCl (pH 7.8) containing 0.5% Triton X-100 and 0.6 M KCl at room temperature for 1 hour and then diluted in phosphate-buffered saline (PBS). After incubation of virus-coated plates with test serum samples for 1 hour, bound antibody was detected with a rabbit anti-mouse IgA (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) and a goat anti-mouse IgG (Boehringer, Mannheim, Germany) conjugated to horseradish peroxidase. Neutralizing antibody titers in serum samples of the immunized mice were also evaluated. The sera were treated with receptor-destroying enzyme (Accurate Chemical and Scientific Corp.) to destroy inhibitors of influenza virus replication. After inactivation of the receptor-destroying enzyme by treatment at 56° C. for 30 minutes, VN1203 and A/Indonesia/7/05 viruses were each incubated with twofold serial dilutions of serum (starting at a 1:10 dilution) at 37° C. for 1 hour. Viral infectivity was determined by titration of the samples in a plaque assay on MDCK cells. The neutralizing titer was defined as the reciprocal titer of serum required to neutralize at least 50% of each virus.

Results

In vitro growth properties of VN1203 viruses possessing M2 cytoplasmic tail deletion mutations. A series of M2 cytoplasmic tail deletion mutants of a highly pathogenic H5N1 (VN1203) virus was generated by reverse genetics as described in Neumann et al. (1999). Transfectant viruses were harvested at 48 hours posttransfection and used to inoculate M2CK cells to propagate stock viruses. The stock virus titers were comparable to that of the wild-type virus: 6.2×10⁸ PFU/mL for VN1203M2del44, 6.8×10⁸ PFU/mL for VN1203M2del33, 6.3×10⁸ PFU/mL for VN1203M2del22, 5.4×10⁸ PFU/mL for VN1203M2dl11, 6.1×10⁸ PFU/mL for VN1203M2del5, and 2.4×10⁸ PFU/mL for the wild-type virus. The only exception was VN1203delM2 (6.0×10⁶ PFU/mL).

Next, the growth properties of the VN1203 M2 tail mutant viruses were compared with those of wild-type VN1203 virus in MDCK cells (FIG. 2). MDCK cells were infected with viruses at an MOI of 0.001, and their growth kinetics were monitored for 72 hours. The VN1203M2del5 and -M2del11 viruses grew as well as the wild-type virus. By contrast, the VN1203M2del22, -M2del33, and -M2del44 viruses replicated less efficiently than the wild-type virus (1,000 to 10,000-fold-lower growth). In particular, the VN1203delM2 virus, which lacks both the TM and cytoplasmic tail domains, was significantly growth restricted on MDCK cells (100,000-fold-lower growth than the wild-type virus). These results are consistent with previous findings that mutant viruses with deletions at the C-terminus of the M2 tail grew less well in cell culture (Itwasuki-Horimoto et al., 2006; McCown et al., 2006; McCown et al., 2005).

In vivo growth properties of VN1203 M2 tail deletion mutants. To determine the virulence of the M2 tail mutants, their growth properties in mice were examined. Mice were infected with 100 PFU of M2 mutant or wild-type viruses. On day 3 postinfection, organs were taken from the infected mice for virus titration. As shown in Table 1, the wild-type VN1203 virus replicated well in all organs examined. Mutants possessing deletions of more than 22 amino acids were not recovered from any of the infected mice. Of interest, replication of the VN1203M2del5 and -M2del11 viruses was more than 1 log lower in the lungs, 2 logs lower in nasal turbinates, and 2 logs lower in the kidneys of infected mice than that of wild-type virus. Moreover, no virus was detected from the brain samples of mice infected with the VN1203M2del11 virus. These results indicate that the VN1203M2del11 virus was attenuated in mice, despite replicating as well as the wild-type virus in MDCK cells.

TABLE 1 Replication of M2 mutant viruses in mice Virus titer (mean log₁₀ PFU/g ± SD) in^(a): Virus Lungs Nasal turbinates Brains Spleens Kidneys Wild-type 8.41 ± 0.09 6.66 ± 0.85 5.02 ± 1.56 7.48 ± 0.48 6.23 ± 0.82 VN1203M2del5 7.47 ± 0.29 4.70 ± 1.21 3.60, 3.51 5.54 ± 0.85 3.90, 4.03 VN1203M2del11 7.30 ± 0.45 4.06, 4.74  ND^(b) 3.97 ± 0.81 4.24 VN1203M2del22 ND ND ND ND ND VN1203M2del33 ND ND ND ND ND VN1203M2del44 ND ND ND ND ND VN1203delM2 ND ND ND ND ND ^(a)Mice were infected with 100 PFU of M2 mutant or wild-type virus. Organ samples were taken from mice at day 3 postinfection. Virus titers were determined with M2CK cells. When virus was not recovered from all three mice, individual titers were recorded. ^(b)ND, not detected.

Generation of a recombinant VN1203 virus that possesses M2del11 and an avirulent HA. Since the VN1203M2del11 virus was attenuated in mice, the feasibility of using it for an H5N1 vaccine was tested. To improve the safety of an H5N1 virus vaccine, vaccine candidates should have multiple attenuating mutations in the viral genes. Therefore mutations were introduced into the cleavage site of the VN1203M2del11 virus HA, a virulence determinant of influenza viruses in birds and mammals (Hatta et al., 2001; Klenk et al., 1994; Steinhauer et al., 1999). In general, low-pathogenicity viruses do not contain a series of basic amino acids at the HA cleavage site (Klenk et al., 1994; Senne et al., 1996; Steinhauer, 1999), restricting cleavage and viral replication to a limited number of organs (i.e., these viruses cause localized infections). By contrast, the HAs of highly pathogenic H5N1 avian influenza viruses contain a series of basic amino acids at this site (Bosch et al., 1981; Garten et al., 1981; Senne et al., 1996; Suarez et al., 1995), which allow HA to be cleaved not only by trypsin but also by ubiquitous cellular proteases (Horimoto et al., 1994; Stieneke-Grober et al., 1992), thereby allowing viral replication in a variety of organs, including brain (i.e., these viruses cause systemic infections). To ensure the safety of the vaccine strains, a mutant HA was constructed in which the amino acid sequence at the HA cleavage site, PQRERRRKKR/G (SEQ ID NO:13), was converted to the sequence in a typical avirulent avian virus, PQ-RETR/G (dashes indicate deletions; SEQ ID NO:14). A recombinant virus possessing this avirulent HA and M2del11 mutations (designated M2del11-HAavir) was generated. Stock virus was amplified on M2CK cells, and the virus titer was 2.0×10⁶ PFU/mL.

Characterization of the recombinant M2del11-HAavir virus in vitro and in vivo. To characterize the M2del11-HAavir virus, its trypsin dependency in vitro was examined. Plaque assays were performed on M2CK cells in the presence or absence of trypsin. With the M2del11-HAavir virus, clear plaques were visible only in the presence of trypsin, whereas the M2del11 virus formed plaques in both the presence and absence of trypsin (data not shown).

Next, to investigate the virulence of the M2del11-HAavir virus in vivo, mice were infected with various doses of the virus and monitored for 14 days (FIG. 3). Even at a high dose (1×10⁵ PFU), the virus did not kill any mice (the MLD₅₀ was >10⁵ PFU, compared to 2.1 PFU for the wild-type VN1203 virus [data not shown]), although slight weight loss was observed (FIG. 3). Mice infected with 100 or 1,000 PFU of the M2del11-HAavir virus did not show any weight loss. Organ tropisms for the M2del11-HAavir virus in mice were also examined. As shown in Table 2, the virus titers were 1 log lower in the lungs of mice infected with 100 PFU of the M2del11-HAavir virus than in those of mice infected with the wild-type virus. No virus was detected in the other organs of M2del11-HAavir-infected mice. Even in the mice infected with a high dose (1,000 PFU) of M2del11-HAavir, the virus was recovered only from the lungs and nasal turbinates, indicating that virus replication was restricted to the respiratory tracts. These results suggest that the M2del11-HAavir virus was highly attenuated in mice.

TABLE 2 Replication of M2delII-HAavir virus in mice Virus titer (mean log₁₀ PFU/g ± SD) in^(a): Dose Nasal (PFU/mouse) Lungs turbinates Brains Spleens Kidneys 100 6.38 ± 1.28 ND^(b) ND ND ND 1,000 6.77 ± 0.17 3.67 ± 1.25 ND ND ND ^(a)Mice were infected with 100 or 1,000 PFU of M2 del11-HAair virus. Organ samples were taken from mice at day 3 postinfection. Virus titers were determined with M2CK cells. ^(b)ND, not detected.

Antibody responses of mice immunized with the M2del11-HAavir virus. To test the efficacy of the M2del11-HAavir virus as a vaccine, mice were intranasally administered with 100 or 1,000 PFU of the M2del11-HAavir virus. Three weeks later, IgG and IgA levels in sera, trachea-lung washes, and nasal washes of immunized mice were measured by means of an ELISA (FIG. 4). Both IgG and IgA levels in trachea-lung washes were significantly higher in mice immunized with the M2del11-HAavir virus than in those treated with a PBS control, although there was no significant difference between the antibody titers in nasal washes from the vaccine group and the control group. The IgA response was negligible in serum, regardless of the dose of the mutant virus used for immunization, but IgG production was clearly higher in mice inoculated with the M2del11-HAavir virus. These data suggest that the M2del11-HAavir virus elicited a significant antibody response in the immunized mice.

To examine whether or not the antibodies detected by ELISA contribute to neutralization of the H5N1 virus infectivity, the infectivity-neutralizing activity of the samples against VN1203 (homologous virus; Glade 1) and A/Indonesia/7/2005 (Indonesia 7) (heterologous virus; Glade 2), whose HA homology is 96.5% at the amino acid level was tested. Immunization with 1,000 PFU of M2del11-HAavir virus did not elicit neutralizing antibody efficiently, and the reciprocal titers of serum required to neutralize 50% of VN1203 and Indonesia 7 were only 31 and 23, respectively (data not shown). Moreover, no neutralizing antibody was detectable in sera from mice immunized with 100 PFU of M2del11-HAavir virus (data not shown), indicating that only a limited level of neutralizing antibody was elicited upon immunization of mice with the M2del11-HAavir virus despite high levels of protection upon lethal challenge and high levels of IgG detected by ELISA.

Protective efficacy of the M2del1-HAavir virus in mice. Mice immunized with the M2del11-HAavir virus were challenged 1 month after immunization with 100 MLD₅₀ of the wild-type VN1203 virus (Glade 1) or Indonesia 7 (Glade 2). Unlike control mice, all M2del11-HAavir-immunized mice survived a lethal challenge with either of the highly pathogenic H5N1 viruses (data not shown) and did not show any symptoms, including weight loss, after the challenge. By contrast, all of the control mice died or had to be euthanized due to their symptoms by day 8 postchallenge (data not shown). The virus titers in several organs of the mice challenged with the VN1203 or Indonesia 7 virus (Table 3) was also determined. High titers of viruses were recovered from all organs of the control group. No virus was detected from any of the organs in the M2del11-HA virus vaccine group challenged with VN1203, though a limited amount of virus was detected in the nasal turbinates of one of the immunized mice challenged with the Indonesia 7 virus (Table 2). Taking the results together, it was concluded that the M2del11-HAavir virus can confer protective immunity to mice against lethal challenge with highly pathogenic H5N1 virus.

TABLE 3 Replication of M2 mutant viruses in mice Challenge Virus titer (mean log₅₀ PFU/g ± SD) in^(a): Virus Group Lungs Nasal turbinates Brains Spleens Kidneys VN1203 PBS 7.83 ± 0.46 6.11, 4.19 3.04 4.96 ± 0.66 2.78, 4.27 M2del11-HAavir  100 PFU  ND^(b) ND  ND^(b) ND ND 1,000 PFU ND ND ND ND ND Indonesia 7 PBS M2del11-HAavir 9.06 ± 0.10 7.01 ± 0.21 3.32 ± 1.37 5.64 ± 0.12 4.27 ± 0.38  100 PFU ND ND ND ND ND 1,000 PFU ND 1.96 ND ND ND ^(a)Three mice from each group were sacrificed on day 3 postchallenge for virus titration. When virus was not recovered from all three mice, individual titers were recorded. ^(b)ND, not detected.

DISCUSSION

The influenza A virus M2 is a multifunctional protein. It has ion channel activity in its TM domain (Pinto et al., 1982), which is thought to function at an early stage of replication (acidification of the virion interior) (Helenius, 1992; Martin et al., 1991; Sugrue et al., 1991) and at a late stage (protection of an acid-mediated conformational change of cleaved HA) (Hay et al., 1985; Ohuchi et al., 1994; Takeuchi et al., 1991). In addition, its cytoplasmic tail is important for viral assembly (Itwasuki-Horimoto et al., 2006; McCown et al., 2006; McCown et al., 2005). In this study, a series of M2 tail deletion mutants was generated and their growth properties in vitro and in vivo examined. Deletions of 5 or 11 amino acids from the C-terminus of M2 were found to not affect virus replication in cell culture but inhibited virus growth in mice. Previously it was shown that even one amino acid deletion from the M2 C-terminus attenuated influenza virus in ferrets (Castrucci et al., 1995). Those findings indicate that the M2 cytoplasmic tail has a vital role(s) in virus replication in animals and that M2 tail mutants could be good vaccine candidates for influenza virus infection. Here, it was demonstrated that H5N1 M2del11-HAavir virus, which has an 11-amino-acid deletion from the C-terminus of its M2 protein and an avirulent HA, protected mice from a lethal challenge with H5N1 viruses, indicating its considerable potential as a live virus vaccine against highly pathogenic H5N1 viruses.

Recently, Suguitan et al. (2006) tested the vaccine efficacy in mice and ferrets of live attenuated, cold-adapted virus vaccine candidates that possess the modified avirulent type of HA and the NA from H5N1 strains, together with the internal genes from cold-adapted A/Ann Arbor/6/60 (H2N2). They demonstrated that a single dose of the vaccine protected animals from lethality but did not fully protected them from replication of the challenge H5N1 viruses, indicating limited efficacy for single-dose vaccination of these cold-adapted viruses. This incomplete protection may stem from unmatched antigenicity between the internal proteins of the cold-adapted virus (i.e., derived from H2N2 virus) and the challenge virus. Here, it was shown that the M2del11-HAavir virus, whose eight genes are derived from an H5N1 virus, protects mice almost completely from replication of heterologous H5N1 virus as well as homologous virus (Table 7). Despite its complete protection, the M2del11-HAavir virus did not elicit neutralizing antibody against either homologous or heterologous viruses efficiently, whereas it elicited high levels of antibodies detected by ELISA. Therefore, cytotoxic T-lymphocyte responses specific to viral internal proteins that contain common cytotoxic T-lymphocyte epitopes among influenza A viruses (i.e., NP and M proteins) and mucosal immune responses may be responsible for the cross-protection observed in this study, as suggested in Takeda et al. (2003). If a vaccine against pandemic influenza is introduced only once a pandemic is imminent, all of the eight genes of the vaccine candidates could be derived from the pandemic strain to offer optimal protection to humans from virus infection. To reduce the risk of the emergence of the revertants, live attenuated virus vaccines should have multiple attenuating mutations in the genes that encode their internal proteins. NS1 mutant viruses are highly attenuated in mice because they lack interferon antagonist activity while retaining the ability to induce protective immunity against influenza virus challenge (Talon et al., 2000). Hence, by combining a mutant NS1 protein with the M2 tail deletion mutants identified in this study, an improved “master” influenza virus could be produced as a first step in the production of safe live influenza vaccines. Continued progress in understanding the functions of these influenza virus proteins should allow the introduction of multiple mutations in live vaccine strains, in addition to those in the HA, NS, and M genes, thereby reducing the likelihood of the emergence of pathogenic revertant viruses.

For live attenuated H5N1 virus vaccines to be clinically useful, the binding specificity of H5 HA for α-2,3-linked sialic acid (SA) receptors, which are preferentially recognized by avian influenza virus and rarely present in the upper respiratory tract of humans (Conner et al., 1994; Rogers et al., 1989; Rogers et al., 1983), must be considered. To address this problem, one could modify the H5 HA to alter its specificity for SA receptors. Recently, Auewarakul et al. (2007); Yamada et al. (2006); Yang et al. (2007) have determined specific amino acids in the avian H5 HA that alter its receptor-binding specificity toward α-2,6-SA (human-type receptor) recognition. This strategy may allow the generation of a recombinant H5N1-based vaccine that recognizes human-type α-2,6-SA receptors and efficiently replicates in the upper respiratory tract in humans.

Example 2

Several lines of evidence suggest that the M2 protein of influenza A virus is responsible for key steps in the viral life cycle (Hay et al., 1985; Iwatsuki-Horimoto et al., 2006; Martin et al., 1991; McCown et al., 2005; Ohuchi et al., 1994; Sugrue et al., 1991; Takeuchi et al., 1994). Indeed, it has previously been shown that the influenza A virus that lacks the TM and cytoplasmic tail domains (M2 knockout [M2KO]) of M2 is highly attenuated in cell culture and in mice compared to the wild-type virus (Iwatsuki-Horimoto et al., 2000; Watanabe et al., 2001). The potential of M2KO influenza A virus as a live attenuated vaccine was examined by immunizing mice and testing immune responses and protective efficacy in a mouse model experimentally infected with A/Puerto Rico/8/34 (PR8), a highly lethal virus in mice.

To generate the M2KO virus, two stop codons were inserted in the M gene segment downstream of the open reading frame of the M1 protein to remove the TM and cytoplasmic tail domains of the M2 protein (FIG. 7A). Then, wild-type PR8 and M2KO viruses were generated by plasmid-based reverse genetics (Neumann et al., 1999). To confirm attenuation of the M2KO virus, the PR8 and M2KO viruses were inoculated into both MDCK and M2CK cells (Iwatsuki-Horimoto et al., 2001) at a multiplicity of infection of 0.001, and virus titers were determined at various times postinfection by using M2CK cells (FIG. 7B). M2KO virus was highly attenuated in MDCK cells (FIG. 7B, left) but replicated as well as the wild-type virus in M2CK cells (FIG. 7B, right). These data suggest that the M2KO virus is highly attenuated in normal cells but that high titer virus stocks can be produced in cells expressing the M2 protein.

Attenuation of viruses in animals is essential for live vaccines. The pathogenicity of the M2KO virus was examined in mice. 4-week-old female BALB/c mice were intranasally infected with different doses of M2KO virus and the virus titers in lungs and nasal turbinates determined. Body weights were also monitored. When mice were infected with even 3×10⁶ or 3×10⁵ PFU of virus, virus was recovered from the lungs, but titers were significantly lower than those in the lungs of mice infected with PR8 (P<0.05) (Table 4). By day 8 postinfection, M2KO virus was no longer detected in the lungs (data not shown). Although virus was recovered from the lungs of one of the animals infected with 3×10⁴ PFU of virus, virus was not detected from any mice infected with lower titers (i.e., 3×10² or 3×10³ PFU) of M2KO virus (Table 4 and data not shown). In nasal turbinates, no virus was recovered from any mice inoculated with the M2KO virus on days 3 and 6 postinfection (Table 4). The body weights of mice infected with 5×10⁶ PFU of PR8 rapidly decreased, and these mice were euthanized by 4 days postinfection (FIG. 7C). On the other hand, mice infected with 3×10⁶ PFU of M2KO virus showed no body weight loss (FIG. 7C). Taken together, these data indicate that the M2KO virus is highly attenuated in mice, satisfying its requirement for a live attenuated influenza vaccine.

TABLE 4 Replication of M2KO virus in mice^(a) Virus titer (mean ± SD log₁₀ [PFU/g]) from PFU of virus indicated source^(b) inoculated per Days Nasal Virus mouse postinfection Lungs turbinates PR8 1 × 10³ 3 8.5 ± 0.1 6.0, 5.8 1 × 10³ 6 7.0 ± 0.1 6.4 ± 0.2{grave over ( )} M2KO 3 × 10⁶ 3 5.7 ± 0.3 ND 3 × 10⁶ 6 5.5 ± 0.3 ND 3 × 10⁵ 3 4.1 ± 0.2 ND 3 × 10⁵ 6 5.3, 4.2 ND 3 × 10⁴ 3 5.2 ND 3 × 10⁴ 6 ND ND ^(a)Three BALB/c mice per group were intranasally infected with the indicated amounts of virus (50 μL per mouse) and sacrificed on days 3 and 6 prostinfection for virus titration. When virus was not recovered from all three mice, individual titers were recorded. On day 3 postinfection, virus was not recovered from organs of mice infected with either 3 × 10² or 3 × 10³ PFU of M2KO virus (data not shown). ^(b)ND, not detected (detection limit, 10 PFU/mL/lung).

The level of antibody responses elicited by the M2KO virus was also determined. 4-week-old female BALB/c mice were intranasally inoculated with different doses of M2KO virus. As negative and positive controls, mice were also intranasally inoculated with phosphate-buffered saline (PBS) or a dose equivalent to 3×10⁶ PFU of formalin-inactivated PR8 (32 hemagglutination units), and a 50% mouse lethal dose of PR8 of 0.3 (500 PFU), respectively. Four weeks after inoculation, titers of immunoglobulin G (IgG) and IgA antibodies against PR8 in sera, trachea/lungs, and nasal washes were determined by an enzyme-linked immunosorbent assay (FIG. 8). Neither the IgG nor the IgA response was appreciable in negative control mice (PBS and inactivated PR8). Although IgG and IgA titers in mice infected with a 50% mouse lethal dose of PR8 of 0.3 were higher, those in mice inoculated with 3×10⁶ PFU of M2KO virus were also similarly increased. Moreover, antibody responses correlated with the doses of M2KO virus, although responses in mice inoculated with 3×10² PFU of M2KO virus were limited.

To assess the protective efficacy of M2KO virus, mice intranasally inoculated with M2KO virus, formalin-inactivated PR8, or PBS were challenged with a lethal dose of PR8 at 4 and 12 weeks postimmunization. Virus titers in lungs and nasal turbinates of challenged mice were determined by using MDCK cells 3 days postchallenge. Virus could not be detected in organs of mice inoculated either with 3×10⁶ or 3×10⁵ PFU of M2KO viruses, indicating that vaccination with these amounts of M2KO virus gave mice sterile immunity (Table 5). All mice immunized with lower doses (3×10⁴ and 3×10³ PFU), with the exception of one mouse inoculated with 3×10³ PFU of virus, survived a lethal challenge, although virus was detected in both organs tested and the amounts of virus were not significantly lower than those in the control mice (Table 2). This is probably because virus clearance later than day 3 postchallenge (the time point at which organ virus titers were examined) was more efficient in these immunized mice than that in the control mice. Indeed, although these mice lost body weight until 7 days postchallenge, they ultimately regained their body weight (data not shown). Taken together, the data indicate that M2KO virus confers effective protection against challenge with a lethal dose of PR8.

TABLE 5 Protection against challenge with lethal doses of PR8 in mice immunized with M2KO virus^(a) Virus titer (mean ± SD No. of log₁₀ ([PFU/g]) from Weeks PFU of virus survivors/ indicated source^(c) postimmu- Immu- inoculated no. of mice Nasal nization nogen per mouse tested Lungs turbinates 4 M2KO 3 × 10⁶ 8/8 ND ND M2KO 3 × 10⁵ 8/8 ND ND M2KO 3 × 10⁴ 8/8 6/3 ± 1.5 5.1 ± 0.3 M2KO 3 × 10³ 7/8 6.9, 6.6 6.2, 6.0 M2KO 3 × 10² 0/8 8.0 ± 0.1 6.3 ± 0.3 Inacti- 0/8 7.6 ± 0.1 5.9 ± 0.2 vated PR8^(b) PBS 0/8 7.5 ± 0.1 5.9 ± 0.1 12 M2KO 3 × 10⁶ 8/8 ND ND M2KO 3 × 10⁵ 8/8 ND ND M2KO 3 × 10⁴ 8/8 7.2 ± 0.6 6.0 ± 0.3 M2KO 3 × 10³ 7/8 7.4 ± 0.6 6.3 ± 0.2 M2KO 3 × 10² 0/8 8.5 ± 0.2 6.9 ± 0.2 Inacti- 0/8 7.3 ± 0.2 6.7 ± 0.3 vated PR8 PBS 0/8 7.8 ± 0.3 6.2 ± 0.6 ^(a)Twenty-two BALB/c mice per group were intranasally immunized with the indicated amounts of M2KO virus, inactivated PR8, or PBS (50 μl per mouse). Half of the mice were challenged with a 50% lethal dose of wild-type PR8 of 100, 4 weeks postimmunization, and the remaining mice were challenged 12 weeks postimmunization. Eight mice per group were monitored for survival for 14 days after challenge. Three mice per each group were sacrificed on day 3 postchallenge to measure virus titration. When virus was not recovered from all three mice, individual titers were recorded. ^(b)Virus particles equivalent to 3 × 10⁶ PFU (32 hemagglutination units) were used. ^(c)ND, not detected (detection limit, 10 PFU/mL/lung).

Thus, M2KO influenza virus that lacks the TM and cytoplasmic tail domains of the M2 protein can be used as a live attenuated influenza vaccine. It appears to be considerably safe and strongly immunogenic. Moreover, the growth profile of this virus is indistinguishable from that of the parent virus (PR8) in cells stably expressing the wild-type M2 protein, suggesting the feasibility of efficient vaccine production, although the cell line expressing the M2 protein may need to be validated for the lack of unwanted properties, such as the presence of adventitious agents and tumorigenicity, prior to its use in vaccine production for humans.

Embryonated hen eggs are currently used for the manufacture of the vaccine; however, they have potentially serious problems. One of the major problems is that cultivation of viruses in eggs can lead to the selection of variants that are antigenically distinct from viruses grown in mammalian cells (Katz et al., 1987; Robinson et al., 1985; Schild et al., 1983). In addition, there is a risk of allergic sensitization and reactions to egg proteins present in vaccines made from embryonated eggs. However, isolation of human influenza viruses from mammalian cells allows one to obtain viruses that are closely related to those present in clinical specimens of influenza patients (Katz et al, 1990; Robertson et al., 1990; Robertson et al., 1991). In addition, it has been demonstrated that inactivated vaccines prepared from cell-grown viruses induce greater cross-reactive serum antibody and cellular responses or protect better than those made from egg-grown viruses compared in animal models (Alymora et al., 1998; Bruhl et al, 2000; Katz et al., 1989; Wood et al., 1989). The fact that M2KO virus amplification relies on cells stably expressing the wild-type M2 protein demonstrates its suitability for vaccine production and solves many of the problems associated with vaccines made from embryonated eggs.

There has always been a question regarding the safety of live influenza vaccines due to possible reassortment between field strains and attenuated vaccine strains during epidemics and pandemics. However, such concerns are unfounded because even if reassortment occurs, as long as the backbone virus for the live vaccines is less pathogenic than the field strains, the pathogenicity of the resultant reassortant would be the same or less than that of the field strain.

Recent outbreaks of highly pathogenic H5N1 avian influenza virus pose a serious threat to human health. Although several clinical trials using inactivated H5 vaccine have been conducted (Bressen et al., 2006; Lin et al., 2006; Nicholson et al., 2001; Stephanson et al., 2003; Treanor et al., 2006), the efficacy of inactivated vaccines against such highly pathogenic viruses in immunologically naïve populations remains unknown. It is important to explore live vaccines, which could provide better protective immunity than inactivated vaccines due to their ability to provide mucosal and cellular immune responses. The M2KO vaccine disclosed here, together with the M2 cytoplasmic tail mutant vaccine that was recently reported (Watanabe et al., 2008), may set the stage for further development of live attenuated H5 influenza vaccines.

REFERENCES

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All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention. 

1. A vaccine comprising an effective amount of an isolated recombinant influenza virus comprising a mutant M gene segment that is mutated so that upon viral replication the mutant M gene expresses a functional M1 protein and a mutant M2 protein with a deletion of the cytoplasmic tail and either lacking a transmembrane domain or having a mutated transmembrane domain, wherein the replication of the recombinant virus is attenuated in vivo relative to a corresponding influenza virus with a wild-type M gene segment.
 2. The vaccine of claim 1 wherein the mutant M2 protein comprises the M2 extracellular domain.
 3. The vaccine of claim 2 wherein the M2 extracellular domain comprises less than 24 residues.
 4. The vaccine of claim 2 wherein the M2 extracellular domain comprises at least 9 residues.
 5. The vaccine of claim 1 wherein the mutant M2 protein in the recombinant virus further comprises a heterologous protein at the C-terminus of the extracellular domain.
 6. The vaccine of claim 1 wherein the mutation in the transmembrane domain comprises at least one amino acid substitution.
 7. The vaccine of claim 1 wherein the mutation in the transmembrane domain comprises a deletion in the transmembrane domain.
 8. The vaccine of claim 7 wherein the deletion in the transmembrane domain includes residues 29 to
 31. 9. The vaccine of claim 7 wherein the deletion in the transmembrane domain comprises at least 10 residues.
 10. The vaccine of claim 1 wherein the recombinant virus further comprises a heterologous immunogenic protein of a pathogen.
 11. The vaccine of claim 1 wherein the recombinant virus comprises influenza A HA.
 12. The vaccine of claim 11 which comprises H5 HA.
 13. The vaccine of claim 11 wherein the HA is not H1 or not H3.
 14. The vaccine of claim 11 wherein the HA in the recombinant virus is modified at the HA cleavage site.
 15. The vaccine of claim 1 wherein the recombinant virus further comprises another attenuating mutation.
 16. The vaccine of claim 1 further comprising a different influenza virus.
 17. The vaccine of claim 1 further comprising two different influenza viruses.
 18. The vaccine of claim 1 wherein the recombinant influenza virus is a reassortant virus.
 19. The vaccine of claim 18 where the reassortant comprises at least six gene segments from a master vaccine strain.
 20. A method to immunize a vertebrate, comprising: contacting the vertebrate with the vaccine of claim
 1. 21. The method of claim 20 wherein the vertebrate is an avian.
 22. The method of claim 20 wherein the vertebrate is a mammal.
 23. The method of claim 20 wherein the vertebrate is a human.
 24. The method of claim 20 wherein the HA is H5 HA.
 25. The method of claim 20 wherein the HA is H1 HA. 